Preparation of insulin



United States Patent PREPARATION or INSULIN Loyal C, Maxwell, Chicago, and William P. Hinkel, Brookfield, Ill., assignors to Armour and company, ChicagoJlL, a corporation of lllinois No Drawing. Application April "3, 1951', Serial No. 219,125

The portion of the-term of the patent subsequent'to' May 6, 1969, has been-dis'claimed 4 Claims; (Cl; 167-45) This invention relates to the preparation ofi'insulin from pancreas'glands; This application constitutes a continuati0n-in-part of our co-pendingapplications;Serial Nos. 103,956 filed July 9, 1949, now-abandoned, and 158,928, now Patent 2,595,278.

In the commercialmanufacture of insulin, the practice has been to extract the insulin from pancreas glands, using acidified alcoholic solutions employing hydrochloric acidor sulphuric acid. of the extraction mixture is normally; maintainedat about 2.0.' The pancreas glands are bashed" and,- in -theusual commercial process, the insulin is extracted' therefrom by usin'ga solvent consisting.'of-'-ethyl alcohol-and hydro-' chloric acid, the alcohol being; in a concentration: 'of approximately 65% to 75% on the'basisiofatheextraction volume after equilibriurn' is attained.v After adjusting the pH to about 2.0; the mixture isuagitated for-several'hours and centrifuged, the meat solids being reextractedunder the same conditions. The centrifuga'te liquors from both steps are then made alkaline; usingw-ammonia, andare filtered. The filteredextracts are concentrated'and'further purified for insulin recovery.

The foregoing process gives a yield which amounts to only a small percentageof'the insulimwebelievedto be actually contained in the glands; Since the amount of insulin available to diabetic-patients is critical, the matter of yield is one of great'importan'ce, and weset' about to discover some method which will give greater yields of insulin in the extraction process and further yields of the final finished product in crystalline or. amorphous'con dition for clinicaladministration. We have'found that it is possible to employ phosphoric acid in the extraction step and obtain a substantially increased yield of'insulin from pancreas glands.

We have further found that phosphoricacid, in the extracting solution, hastheuniqueproperty offorrning insoluble salts with bases, permitting-the: remov'alof the phosphoric acid as saltin the filtration operation, and thus greatly increasing the yield of insulin.-

In addition to the use of sulphuric acid and hydrochloric acidas mentioned above," organic acidsysuchasacetic, formic and the like, maybe employed; These "organic acids have to be-usedin such quantities that their use is not considered practicable. Further; the'presence ofthe large amount of solublesalts ofltheseacids'are. undesirable in the subsequent steps ofthe process.

We have found that phosphoric acid,= unlike sulphuric acid, hydrochloric acid and the above organic. acids, has

the unique property offorming.insolubleesalts, thus per-' mittingzthe rem-ovalof the phosphoric'acid from the proc-' ess in an early filtration step followin'g the extraction step. An objectof theinverition is t'o'providea process m which phosphoric acidis employed-in the'extraction step and subsequently removed frorn the process as an insoluble salt. Another object is to provide aprocess' in whichphosphoric acid is. employed'in- 'the extraction step and in which a base is added"for the precipitatiorbof the acid as an insoluble-salt; the'salt being removed along with precipitated protein material: is to providers. processin'whiclr'pliosphoric acid is effec tivel'y employed for the extract-ion of insulin, while at the same time removin'gthe phosphoridacid as a saltto prevent insulin, lossesi in subsequent necessary treating steps; Yetanother object is"to"prov'ideia' process fol-the extraction of' insulin in which phosphoric acidis present In suchextractionsteps, the pH A still further object'- lipoidal material, etc.

2,695 ,8 61 Patented Nov. 30, 1954 in' the extraction step and eliminated as an insoluble salt prior to subsequent treating steps for the removal of Other specific objects and advantages' will appear as the specification proceeds.

In-applicants process, pancreasglandsfrom any source may be employed. For 'example, beef-pancreas glands, pork pancreas? glands and the pancreas glands from other animals and from fish, whale and other sourcesmay' be employed.

In one embodiment of our process, we extract the hashed pancreas-glands in-anorganic' solvent for insulin, such as ethyl alcohol, and in the presence of phosphoric acid; Preferably, sufiicient phosphoric acid isemployed to adjust the pH of the extraction mixture to about 3. The extraction mixture maythe'n be filtered or centrifuged to remove the suspended solids. If desired, the solids or residuemay be reextraeted under substantially the same conditions and the filtrate combined with the first filtrate.

After the extraction step, we prefer to add a base, such as aqueous ammonia, to bring the pH to about 5.58.5, thereby forming an insoluble ammonium salt of the phosphoric acid, while at the same time precipitating inert protein material. The salt and the inert protein material may then be removed'by filtration. Any suitable base for formingan insoluble saltwith the phosphoric acid may be employed. For example, sodium hydroxide may be employed in lieu of'ammonia. We prefer to employ a weak base, and aqueous ammonia-is particularly effective in the operation.

The insulin in the alcoholic filtrates may be recovered by conventional procedures. The alkaline alcoholic filtrates are acidified and'concentrated in vacuo at reduced temperatures and the insoluble lipoidal material removed by' filtration. The insulin in the aqueous concentrate may be precipitated by the addition of sodium chloride, and the salt cake thus obtained may be further purified by solubilizing in water and precipitating the insulin at its iso-electric point. The recovered precipitated insulin may besolubilized and then crystallized after the addition thereto of zinc acetate or zinc chloride, to thereby obtain the zinc insulin salt;

Instead of the conventional purification method just described, it will be understood that other well-known purification methods leading to the preparation of finished amgrphous or crystalline insulin compositions may be uS We prefer to remove lipoidal material in the filtrate after the above filtration stepin which the insoluble salt and inert protein is removed and prior to the purification of the filtrate to obtain insulin. After the filtration step, the filtrate obtained is adjusted by the addition of sulphuric acid (or some other acid) to a pH of 33.5 and concentrated under reduced pressure to an alcohol contentof 15-25%, preferably about 20%. The lipoidal material, which then separates, may be removed by filtration or centrifugation, and the filtrate concentrated again under reduced pressure to the aqueous phase. There is a further precipitation of lipoidal material, and this is then removed by filtration or centrifugation.

The lipoidal material is preferably separated by partial removal of the alcohol under'vacuum in a still. The temperature in this step is preferably kept as low as possible, while the still is maintained under reduced pressure. We have obtained good results by maintaining temperaturesof 60-70 F.

In the removalof lipoidalmaterials, we' have found that an exceptionally sharp and quick separation can be carried out by heating the 15-25% concentrate to a temperature of about to*l10 F. before attempting to separate the lipoidal material. Although the concentration of the alcohol content to' 15 to 25% itself tends to render the lipoids insoluble, the actual separation step at this-point is ordinarily a slow and tedious'one, requiring a lengthy filtration, or combination of filtration and centrifugation operations- Especially when large volumes of materials are being processed, the necessity of holding the extracts during .ext'ended separation steps is costly and tends 'to bringabout a reduction in yield. However, we have found that the operation at this point may be greatly facilitated by taking the 15 to 25% concentrate, which is maintained at a temperature of about 60 to 70 F. during the concentration step, and heating it to about 85 to 110 F. This causes a very rapid flocculation of dispersed fat which rapidly rises and floats on the surface of the extracts, from whichit can readily be removed and discarded. If the resulting extract remains slightly turbid, we have found further that the turbidity can quickly be removed to provide a sparkling clear solution by chilling to a temperature of about 55 to 70 F. and filtering. By the above improved procedure we. have found it usually unnecessary to filter after complete concentration to the aqueous phase, since the lipoidal material is essentially completely removed in the previous step.

In the precipitation of inert proteins, it is advantageous to adjust the pH into the range of 5.2 to about 9.5, with the preferred range being 5 .58.5. Our best results have been obtained in the range of pH 8-8.5. The inert protein material is effectively precipitated, and the removal of this material, together with the insoluble salts of phosphoric acid, has a highly beneficial effect upon the subsequent operations.

The solvent for the insulin may be any water-miscible organic solvent for insulin, such as ethyl alcohol, methyl alcohol, propyl alcohol, isopropyl alcohol, acetone, etc., or mixtures thereof. We prefer to use an aliphatic alcohol of less than 4 carbon atoms, and find ethyl alcohol the most desirable.

The concentration of the organic solvent in the extraction mixture should be sufiiciently high to avoid substantial solubility of other substances, such as the pancreatic enzymes, and should not be so high as to produce substantial insolubilization of insulin. We find that an alcohol concentration of to 85% by volume in the liquid present in the extraction mixture is satisfactory.

Concentrations below 50% permit solubility of an undesirable amount of enzymes together with protein impurities, and concentrations above 85% produce a decreased yield of insulin. We prefer to 75% alcohol concentration.

When We refer to concentration in this specification and claims, we mean the over-all concentration in the extraction mixture on the basis of the total volume of liquid present and not the concentration of the solvent added to the process.

The temperature at which the extraction is conducted may vary throughout a wide range, but we prefer to conduct the extraction step at temperatures between 0 and 15 C., and most suitably at about 10 C.

As above stated, in the recovery of insulin from pancreas glands, the hashed or macerated pancreatic tissue is extracted ordinarily in an acidified alcoholic solution containing 60% to 75% alcohol. The alcoholic extraction mixture ordinarily is acidified to a pH of about 2.0 with hydrochloric acid or sulphuric acid. Other workers more recently have reported using weak organic acids, such as acetic, formic, propionic and butyric acids, for this purpose.

We have shown that phosphoric acid is particularly suitable as an acidifying agent in the extraction of insulin from pancreatic tissue and that very considerably higher yields of insulin are obtained thereby than are obtained by the use of strong acids, such as hydrochloric or sulphuric acids. In the use of phosphoric acid for this purpose, we have found that maximal insulin recovery is obtained when the extraction mixture is acidified to a pH of from 2.5 to 4.0. We prefer to employ a pH of from 2.5 to 3.5 and find the range of 2.85 to 3.5 particularly satisfactory. We do not find it advantageous to reduce the pH below 2.5 since the yield of insulin is not appreciably increased by the use of more acid. Above pH 4.0 the yield drops off sharply.

We believe that the higher yields of insulin obtained with the use of phosphoric acid to be due to a combination of desirable properties possessed by this acid, which makes it particularly suited for use in the extraction of insulin from pancreatic tissue. This combination of desirable properties consists of the property of phosphoric acid of promoting maximum solubilization and extraction of insulin from pancreatic tissue without the necessity of using the excessively high acidity (pH 2.0) required when using hydrochloric or sulphuric acid, and, secondly, the property that the salts of phosphoric acid are relatively insoluble in the extraction solvent, so that the high salt concentration incurred in the extracts using hydrochloric,

til)

acetic or formic acid or other acids of this type, is avoided.

In the use of acetic, formic or hydrochloric acids in the extraction of insulin, it is necessary, after removing the gross pancreatic tissue after extraction, to neutralize the acidity in order to clarify the extracts by filtration. This is usually performed by adjusting the pH of the extracts to pH 78.5 by the addition of aqueous ammonia. This alkalinization also causes the precipitation and allows the removal of undesirable protein impurities which are precipitated in this pH range.

In the use of hydrochloric, acetic or formic acids, the adjustment of the pH of the extracts to 7.0-8.5 previous to filtration results in the introduction of very large amounts of soluble ammonium salts into the extracts. This is undesirable and does not occur when phosphoric acid is employed to acidify the extraction mixture, since the ammonium salts of phosphoric acid are relatively insoluble in the aqueous-alcoholic insulin extractant and are removed when the extracts are filtered after the pH has been adjusted toward neutrality or to the alkaline side with aqueous ammonia. This avoidance of the introduction of large quantities 'of soluble ammonium salts into the extracts contributes to higher insulin recovery, since the presence of such large amounts of salts raises the boiling point of the extracts during the later concentration in the still to remove alcohol and in this way and otherwise is responsible for large losses of insulin.

The use of phosphoric acid also allows the removal of other undesirable substances from the extracts, such as iron, copper, or heavier metals whose salts may be present in small amounts as salts of the metals which may have been introduced as contaminations in the reagents or materials or from the equipment employed in processing. It is known that the presence of such metallic ions causes insulin losses. The salts of such metals with phosphoric acid are comparatively insoluble in the extractant employed and are removed when the extracts are neutralized with ammonia and filtered. Such metallic ions are not so completely removed when sulphuric, hydrochloric, formic or acetic acids are used, since the salts of these acids are comparatively soluble in the extractant and adjustment of the extracts to the alkaline side does not serve to remove them as completely since the hydroxides of these metals are somewhat more soluble than their phos phate salts under the conditions.

We have found that when weak organic acids, such as acetic or formic are used, that it is necessary to use sufficient of these acids to lower the pH of the extraction mixture to about 3 to 3.5 in order to obtain optimal insulin extraction. These weak organic acids have acid dissociation constant of 1.8 10 to 1.75 l0 and three to ten times the weight of such acids is required as compared to the quantity of phosphoric acid required to lower the extraction mixture proportionally. We consider the use of such weak acids impractical since it considerably increases the cost of processing due to the excessive quantities of acid required. Phosphoric acid is comparatively a stronger acid, having an acid dissociation constant of 1.1 10" and We find this strength of acid particularly suitable for our purposes, and we do not find that acids with a dissociation constant of less than l l0 are suitable from a practical standpoint for insulin extraction.

In adjusting the pH of the centrifuged extracts upward in order to precipitate and remove undesirable proteins and insoluble salts of phosphoric acid, we prefer to use a relatively weak base as an alkalinizing agent in order to avoid local spots of unduly high alkalinity such as may occur using a strong base. We find that aqueous ammonia (ammonium hydroxide) is suitable and we prefer its use. However, various weak or strong bases can be used although it is necessary in order to avoid the introduction of large quantities of soluble salts into the extracts, to use a base which forms relatively insoluble salts with phosphoric acid in the given environment when the pH is adjusted to pH 5.5 to 9.5. In the case of ammonia or sodium hydroxide the precipitation of insoluble ammonium or sodium phosphate starts at about pH 5, the precipitation being more complete with increasing alkalinity. In the case of phosphoric acid the precipitation of ammonium phosphate is about two-thirds complete at pH 6 and about complete at pH 8.0.

Therefore, the precipitation of insoluble salts and of undesirable proteins can be conducted between pH 6.0 and pH 9.5. We, however, prefer to adjust the extracts :an alcohol concentration of into-between.- pH 7.5" and 8.5 beforezfiltrationrsince; a; higher alkalinity is not advantageous and. a"; mi nimum'; amount of the salts of phosphoric acid remains soluble in this range.

= In ourpreferred;process, sufficient of the liquidphosphoric acid is added .to; an aqueous-alcoholicsolution. of between65% and 95% alcohol content, ,so .that after addition of the macerated pancreatic tissue, the pH of the extraction mixture is about 3.0. and the alcohol..-.content about 60% to 65 .%r after equilibrium is .attained. After stirring and allowing sufficient time for extraction, the

meat solids are removed by centrifugation and reextracted in 60%. to 65 alcohol with the addition of sufiicient acid ;to: maintain the same pH and again'centrifuged. The pH .of the centrifuged extracts isadjustedto-pH 6.0 to9.5

with the addition of aqueous ammonia-and .filtered .througha filter press to removeprecipitated insoluble salts and proteins and other suspended material. The

clear filtered extracts are reacidified to a pH of;;about 3.5

with sulphuric acid and furtherprocessed and concentrated and purified according to conventional ;and wellknown procedures.

-In acidifying the alcoholic extraction mixture, wedo not. find it advantageous to'reducethe pH of the extraction mixture below pH 2.5 .since we obtain optimal insulin yields in the range of: pH 2.85 to 3.5 and ;do not appreciably increasethe yield by the use;of;more acid.

The. amount of insulin extracted drops offsharply at a .pH above 4.0. The desired rangeis pH 2.5 to 4.0 and our preferred range is pH 2.85 to 3.5.

Specific examples illustrating our improved. process maybe set out as follows:

Example I proteins and phosphoric acid salts. Thesolids were removed by filtration and sulphuric acid was then added to the filtrate to bring the pH to 3.5. The acidified extracts were then concentrated under reduced pressure to Lipoidal material was removed by filtration and the filtrate concentrated under reduced pressure to the aqueous phase. Lipoidal material was then removed by filtration and the insulin-containing filtrateibiologically assayed for insulinactivity.

The biological assay showed the insulinrecovered to be equivalent to 1425 International Units-for-each pound of pancreas glands processed.

Example II 40 poundsoffrozen pork pancreas glands were hashed and extracted by stirring with 45,500 cc. of 85% alcohol containing 925 cc. of phosphoric acid. The acidity of the extraction mixture was pH 3.0 and the alcohol concentration approximately 65 after. equilibrium .was attained. The pancreatic meatsolids removed were then reextracted by stirring in 45,000 cc. of 65% alcohol. The pH of the combined filtrates was raised to pH 8.5 by the addition. of ammonia to precipitate the-phosphoric acid salts and inert proteins. The solids were removed by filtration and sulphuric acid was then added to the filtrate .to bring the pH to 3.5. The acidified extracts-were then concentrated under reduced pressure to an alcohol concentration of 20%. Lipoidal material was then removed by filtration and the insulin-containing filtrate biologically assayed for insulin activity. The biological assay showed the insulin recovered to be equivalent to 1615 International Units for each pound of pancreas glands processed.

Example III 1,500 pounds of frozen beef pancreas glands were hashed and extracted by the use of phosphoric acid in the extraction step as described in Example I. The mixture was acidified to pH 3.5 with phosphoric acid. After extraction, the suspended solids were removed by filtration in a filter press. The insulin-containing filtrate was then adjusted to pH 8 by the addition of aqueous ammonia, and the precipitated ammonium salts of phosphoric. acid and inert proteins..-were removed: by filtration through a filtenpress. Thetfiltratewas. thentacidifiednto pH 3.5 with sulphuric .acid. The mass of the lipoidal material was removed by distilling under reduced pressure toan alcohol content.of;approximately.:20% followedby filtration and by further concentrationiunder reduced pressure to the aqueous-phase. Theinsulincsin the filtrate was then recovered by the .conventionalr-procedure, using sodium chloride, etc. 'The'yield of crystalline insulin amounted .to 1400 International .Unitsper pound of original glandsused.

Similar quantities of frozen-pork pancreastglandsnwere bashed and extracted as above described inrExampledII and with similar results. The yield of the final insulin product in the crystalline .oramoiphous formrrepresented a large increase over the yields obtained-in processes employing the standardhydrochloric acid-process.

Example IV pounds of frozen beef pancreas glands. were hashed into 118 liters of 82% alcohol containing.-2200;cc..of phosphoric acid and extracted bystirr-ing. ThepH ofthe extraction mixture was 3.1 and the alcohol concentration 65% after equilibrium wasattained. The meatrsolids were removed by centrifugation and reextractedin .100 liters of 65% alcohol. The combined centrifuged extracts were adjusted to pH-8.2 with aqueousammoniaand filtered. The filtered extracts were then reacidifiedtopH 3.5 with the addition of sulphuric .acid and. concentrated in vacuo and filtered to .remove lipoids. Biological assay of the aqueous concentrate showedthednsulin. recovered to,amountrof .1876 units;per-pound.of;glands processed. The insulin in the aqueous concentrate was then salted out by the addition of sodium chloride and the insulin salt cake obtained solubilizedin water, and the insulin precipitated at the iso-electric point. Theinsulin precipitate was then solubilized in water containing .a buffer, and the insulin converted to zinc insulin crystals by the addition ofszinc acetate. The yield of .driedtzinc insulin crystals recovered as determined by vweight and biological assay was 1526.units perpound. of pancreas glands.

Example I V-A 2000 pounds of exceptionally good frozen beef pancreas glands from the sarnelot processed invExample IV were hashed into 600 gallons of 93% alcohol containing 730,000 cc. of hydrochloric acid and extracted by stirring. .The pH of the extraction mixture was.2.0 and the ,alcohol concentration 70%. The meat solids were vremovediby centrifugation and reextracted in 600 gallons of 70% alcohol. The combined centrifuged extracts were adjusted to pH 7.6 by the addition of aqueous ammonia and filtered. The filtered extracts were reacidified to pH 3.5 by the addition of sulphuric acid andconcentrated in vacuo and filtered to remove lipoids. jBiological assay indicated the insulin content of the aqueous. concentrate .to be 1000 units perpound of pancreas glands processed. The insulin was. salted out from the aqueous concentrate and purified and crystallized by the sameprocedure employed in Example IV. The yield of, dried zincinsulin crystals recovered as determined by weight and biological assay amounted to 835 units per pound of pancreas glands.

Example V 40 pounds of hashed frozen pork pancreasglands-were extracted by stirring in 47 liters of 82% alcohol containing 800 cc. of phosphoric acid. The pH of the extraction mixture was 3.1 and the alcohol concentration'65% after equilibrium was attained. The'meat residue was removed by centrifugation and then reextracted in 40 liters of;65% alcohol. The combined centr'ifugates were adjusted to pH 8.5 by the addition of aqueous ammonia and filtered.

A sample of the alcoholic insulin containing filtrates was removed and biologically assayed. The insulin content of the filtrate amounted to 2090 units per pound of pancreas processed.

The filtered extracts were then concentrated in vacuo and filtered to remove lipoids. The insulin content of the aqueous filtered concentrate as shown by biological assay was 1760 units per pound pancreas glands processed.

The filtered extracts were then concentrated in vacuo and filtered to remove lipoids. The insulin content of the aqueous filtered concentrate as shown by biological assay was 1760 units per pound pancreas glands processed.

Example V-A 40 pounds of hashed frozen pork pancreas consisting of a representative sample of the same lot of glands processed in Example V were hashed into 47 liters of 82% alcohol containing 9.2 pounds of liquid formic acid. The pH of the extraction mixture was 3.2 and the alcohol concentration approximately 65%. The meat residue was removed by centrifugation and reextracted in 40 liters of 65% alcohol. The combined centrifuged extracts were then adjusted to pH 8.5 with aqueous ammonia and filtered.

A sample of the filtered alcoholic extracts removed and biologically assayed showed the insulin content of the gxtracts to be 1680 units per pound of glands processe The filtered extracts were then reacidified and concentrated and filtered by a procedure identical to that employed in Example V. The yield of insulin in the aqueous concentrate by biological assay was 870 units per pound.

Example IV and IV-A, and Example V and V-A demonstrate the injurious effect of the presence of large amounts of soluble ammonium salts of such acids as hydrochloric acid and of formic acid, whose ammonium salts are not precipitated and removed in the extraction solvent employed. As observed in Example V and V-A, the insulin loss during concentration and filtration of the extracts is much less when phosphoric acid, whose ammonium salts are insoluble and are removed, is employed.

Example VI In a commercial scale operation, 7000 pounds of frozen beef pancreas glands were hashed and macerated and extracted by stirring in 1860 gallons of 82% alcohol containing 153 liters of phosphoric acid (USP). After equilibrium was attained by stirring, the pH was 2.85 and the alcohol concentration about 65%. The pancreatic tissue insolubles were removed by centrifugation and reextracted under the same conditions. After centrifugation, the combined extracting liquid was alkalinized to pH 8.2 by the addition of aqueous ammonia and filtered. The filtered extracts were reacidified to pH 3.0 by the addition of H2804. The reacidified extracts were then partially concentrated in the vacuum still to about alcohol concentration, and the partially concentrated extracts were heated to 90 F. and allowed to stand 10 minutes. formed on the surface of the extracts. The lower insulin containing extracts were drawn off at the bottom of the still, and the upper fat layer discarded. The slightly turbid extracts were then chilled to 65 F. and filtered with the aid of diatomaceous earth filter aid to give a sparkling clear solution. The clarified solution was concentrated to the aqueous phase and at this stage was found to contain 1800 units of insulin per pound of glands processed.

The foregoing process for the extraction and recovery of insulin from pancreas glands may be varied through wide ranges, many of the steps employed being optional. We prefer to extract with the insulin solvent containing phosphoric acid and to follow this step with the addition of a base to form insoluble salts of the phosphoric acid. After the phosphoric acid has been removed from the process in the form of ammonium salts of phosphoric acid, it is found that the resulting extract may be treated much more effectively for the recovery of insulin therefrom.

While, in the foregoing specification, certain steps of the process have been described in considerable detail for the purpose of illustrating embodiments of the invention, it will be understood that such details may be varied At the end of this time, a layer of fat had F widely by those skilled in the art without departing from the spirit of our invention.

We claim:

1. In a process for obtaining insulin from pancreas glands, the steps of forming an extraction mixture of insulin-bearing pancreas glandular solids and an aqueous organic solvent for insulin, said mixture being acidified to a pH from 2.5 to 4.0 with phosphoric acid and said organic solvent therein being at a concentration favoring the solubilization of insulin over pancreatic enzymes, extracting insulin from said glandular solids into said aqueous organic solvent to form an insulin extract, adding ammonia to the extract to adjust the pH thereof from 5.5 to 8.5 for precipitating ammonium salts of phosphoric acid, and separating the phosphoric salts thus precipitated.

2. In a process for obtaining insulin from pancreas glands, the steps of forming an extraction mixture of insulin-bearing pancreas glandular solids and an aqueous organic solvent for insulin, said mixture being acidified to a pH from 2.5 to 4.0 with phosphoric acid and said organic solvent therein being at a concentration favoring the solubilization of insulin over pancreatic enzymes, extracting insulin from said glandular solids into said aqueous organic solvent to form an insulin extract, separating the insulin extract from the glandular solids, adding ammonia to the extract to adjust the pH thereof from 5.5 to 8.5 for precipitating inert protein solids and ammonium salts of phosphoric acid, and separating from said extract the (protein salts and phosphoric acid salts thus precipitate 3. In a process for obtaining insulin from pancreas glands, the steps of forming an extraction mixture of insulin bearing pancreas glandular solids and aqueous ethyl alcohol, said mixture being acidified to a pH from 2.5 to 4.0 with phosphoric acid and the ethyl alcohol therein providing to of the liquid volume, extracting insulin from said glandular solids into said aqueous ethyl alcohol to form an insulin extract, adding ammonia to the extract to adjust the pH thereof from 5.5 to 8.5 for precipitating ammonium salts of phosphoric acid, and separating the phosphoric acid salts thus precipitated.

4. In a process for obtaining insulin from pancreas glands, the steps of forming an extraction mixture of insulin bearing pancreas glandular solids and aqueous ethyl alcohol, said mixture being acidified to a pH from 2.5 to 4.0 with phosphoric acid and the ethyl alcohol therein providing 50 to 85% of the liquid volume, extracting insulin from said glandular solids into said aqueous ethyl alcohol to form an insulin extract, separating the insulin extract from the glandular solids, adding ammonia to the extract to adjust the pH thereof from 5.5 to 8.5 for precipitating inert protein solids and ammonium salts of phosphoric acid, and separating from said extract the protein solids and phosphoric acid salts thus precipitated.

References Cited in the file of this patent UNITED STATES PATENTS Number Name Date 1,669,328 Dudley May 8, 1928 1,866,569 Kharasch July 12, 1932 2,353,016 Daughenbaugh July 4, 1944 2,466,487 Scott et al. Apr. 5, 1949 2,529,152 Grant Nov. 7, 1950 2,571,126 Frederiksen Oct. 16, 1951 2,595,278 Maxwell et al. May 6, 1952 OTHER REFERENCES Somogyi, J. Biol. Chem, vol. 60, 1924, pp. 34 to 47 and 54 to 57. 

1. IN A PROCESS FOR OBTAINING INSULIN FROM PANCREAS GLANDS, THE STEPS OF FORMING AN EXTRACTION MIXTURE OF INSULIN-BEARING PANCREAS GLANDULAR SOLIDS AND AN AQUEOUS ORGANIC SOLVENT FOR INSULIN, SAID MIXTURE BEING ACIDIFIED TO A PH FROM 2.5 TO 4.0 WITH PHOSPHORIS ACID AND SAID ORGANIC SOLVENT THEREIN BEING AT A CONCENTRATION FAVOURING THE SOLUBILIZATION OF INSULIN OVER PANCREATIC ENZYMES, EXTRACTING INSULIN FROM SAID GLANDULAR SOLIDS INTO SAID AQUEOUS ORGANIC SOLVENT TO FORM AN INSULIN EXTRACT, ADDING AMMONIA TO THE EXTRACT TO ADJUST THE PH THEREOF FROM 5.5 TO 8.5 FOR PRECIPITATING AMMONIUM SALTS OF PHOSPHORIC ACID, AND SEPARATING THE PHOSPHORIC SALTS THUS PRECIPITATED. 